Overview

ELISA: The anti HA diluted 1:70.000 gave an O.D.=1.0 in a 15 minute reaction against peptide conjugated with a different carrier than used for anti peptide purification. HRP conjugated Goat anti rabbit IgG was used and TMB was the substrate.

Applications

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application

Abreviews

Notes

ChIP/Chip

Use at an assay dependent concentration.

IP

Use at an assay dependent concentration.

ELISA

1/200 - 1/500.

WB

1/4000 - 1/10000.

ICC/IF

Use a concentration of 1 - 4 µg/ml.

ICC

Use at an assay dependent concentration.

Flow Cyt

Use at an assay dependent concentration.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

ChIP

Use 3 µg for 25 µg of chromatin.

Target

Relevance

Human influenza hemagglutinin (HA) is a surface glycoprotein required for the infectivity of the human virus. The HA tag is derived from the HA molecule corresponding to amino acids 98-106 has been extensively used as a general epitope tag in expression vectors. Many recombinant proteins have been engineered to express the HA tag, which does not appear to interfere with the bioactivity or the biodistribution of the recombinant protein. This tag facilitates the detection, isolation, and purification of the proteins.

Xenopus embryos were injected with 500 pg pkcα-gfp RNA and co-injected as indicated above the images. Animal Caps were prepared at stage 10 and immunostained as indicated. Nuclei were stained with Hoechst 33258 (blue). Images show representative results from at least two independent experiments with a minimum of six Animal Caps per experiment. Scale bars: 50 µm.

ab9110 was diluted to 4 µg/mg lysate and incubated with a nuclear lysate of HEK293T cells transiently expressing HA-tagged protein and a Protein A matrix for 2 hours a 23°C to achieve immunoprecipitation. 1000 µg of lysate was present in the input.

A HRP-conjugated anti-rabbit HA monoclonal antibody diluted 1/1000 was used for the Western Blot step.

This image was kindly supplied as part of the review submitted by Kasper Fugger. Immunofluorescence staining of U-2 cells expressing HA-tagged protein with ab9110.

ChIP - Anti-HA tag antibody - ChIP Grade (ab9110)

Xenopus laevis oocytes were injected with mRNA for HA-tagged human BORIS. Chromatin was prepared according to the Abcam X-ChIP protocol. Oocytes were fixed with formaldehyde for 10min. The ChIP was performed with 25µg of chromatin, 20µl of Protein A/G sepharose beads, and 3µg of ab9110 (anti-HA, light blue) or, 3µg of ab18337 (anti-Boris, dark blue). A non-specific antibody was used as a control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach).

ab 9110 at a 1/200 dilution staining the mouse olineu cell line (oligodendrocyte precursor cell) by immunocytochemistry. The antibody was incubated with the cells for 30 minutes and then detected using a Cy5 conjugated goat anti-rabbit antibody.

This image is courtesy of an Abreview submitted by Katarina Trajkovic on 15 March 2006